Control of gene expression is fundamental to life and this control usually involves transcriptional regulation. Much of the research on transcriptional regulation in eukaryotes over the past 2 decades has focused on regulatory mechanisms that ultimately affect transcription initiation by Pol II at protein-encoding genes. However, recent analyses of Pol II interactions in vivo using genome-wide mapping techniques such as ChIP- chip reveal that thousands of genes in human and Drosophila have Pol II concentrated at the 5'end. Much of this Pol II appears to have initiated transcription but paused in the promoter proximal region. Hence, a prominent control point in the transcription occurs after transcription initiation. This promoter proximal pausing and its regulation are poorly understood largely because it has gone unrecognized at most genes. The hsp70 gene of Drosophila is one of the few genes where promoter proximal pausing has long been known to occur, and it serves a paradigm for this process. The sequence-specific, DNA-binding proteins, GAGA factor and HSF, function respectively in the establishment and reactivation of the paused Pol II. The negative elongation factors, NELF and DSIF, participate in pausing by associating with Pol II. The positive elongation factor, P- TEFb, is thought to reactivate the paused Pol II by phosphorylating NELF, DSIF and Pol II. Even with the identification of these proteins, the mechanisms by which they control the Pol II are not known. The overall goal of this project is to elucidate mechanisms of promoter proximal pausing and its control. Aim 1 determines how DSIF and NELF cause Pol II to pause and how their activities are regulated by post-translational modifications. Aim 2 determines how GAGA factor and HSF control promoter proximal pausing. It also investigates the role of a novel sequence-specific, DNA-binding protein whose DNA binding site is detected in at the promoter of 20% of genes found to be associated with Pol II. Aim 3 will determine if the regulation of promoter proximal pausing involves kinetic competition between the rate of elongation and the rate at which DSIF and NELF capture the elongating Pol II. A combination of biochemical and in vivo approaches applied to the model organism, Drosophila, uniquely qualifies this project for obtaining significant insight into the mechanism of promoter proximal pausing, thereby having significant impact our fundamental understanding of gene regulation. In addition, NELF, DSIF and P-TEFb have each been linked to human maladies. One subunit of NELF associates with the BRCA1 and has been linked to breast cancer. DSIF and P-TEFb both contribute to transcription of the HIV provirus, and an inhibitor of P-TEFb is in phase 2 clinical trials for cancer therapy. PUBLIC HEALTH RELEVANCE: Transcriptional regulation plays a prominent role in the appropriate expression of genes, and many diseases arise because of defects in gene expression. Recent research has revealed that a mechanism of transcriptional regulation called promoter proximal pausing, once thought to occur at only a handful of genes, actually happens at thousands of genes including ones involved in development and stem cell renewal. Promoter proximal pausing will be studied in the model organism, Drosophila, which offers a unique combination of experimental approaches for understanding this important biological process.